Oxibendazole Research Articles

In Silico Drug Repurposing Uncovered the Antiviral Potential of the Antiparasitic Drug Oxibendazole Against the Chikungunya Virus.

Chikungunya virus (CHIKV) has been reported in over 120 countries and is the causative agent of Chikungunya fever. The debilitating nature of this disease, which can persist months to years after acute infection, drastically impacts the quality of life of patients. Yet, specific antivirals are lacking for the treatment of this disease, which makes the search for new drugs necessary. In this context, the nsP2 protease emerges as an attractive therapeutic target, and drug repurposing strategies have proven to be valuable. Therefore, we combined in silico and in vitro methods to identify known drugs as potential CHIKV nsP2 protease inhibitors with antiviral properties within DrugBank. Herein, we developed a hybrid virtual screening pipeline comprising pharmacophore- and target-based screening, drug-like, and pharmaceutical filtering steps. Six virtual hits were obtained, and two of them, capecitabine (CPB) and oxibendazole (OBZ), were evaluated against CHIKV replication in Vero cells. CPB did not present antiviral activity, whereas OBZ inhibited the replication of two different strains of CHIKV, namely, 181-25 (Asian genotype) and BRA/RJ/18 (clinical isolate from ECSA genotype). OBZ showed potent antiviral activity against the CHIKV BRA/RJ/18 (EC50 = 11.4 μM) with a high selectivity index (>44). Analogs of OBZ (albendazole, fenbendazole, and mebendazole) were also evaluated, but none exhibited anti-CHIKV activity, and further, their stereoelectronic features were analyzed. Additionally, we observed that OBZ acts mainly at post-entry steps. Hence, our results support further in vivo studies to investigate the antiviral potential of OBZ, which offers a new alternative to fight CHIKV infections.

Ascarids exposed: a method for in vitro drug exposure and gene expression analysis of anthelmintic naïve Parascaris spp.

Ascarid parasites infect a variety of hosts and regular anthelmintic treatment is recommended for all species. Parascaris spp. is the only ascarid species with widespread anthelmintic resistance, which allows for the study of resistance mechanisms. The purpose of this study was to establish an in vitro drug exposure protocol for adult anthelmintic-naïve Parascaris spp. and report a preliminary transcriptomic analysis in response to drug exposure. Live worms were harvested from foal necropsies and maintained in RPMI-1640 at 37 °C. Serial dilutions of oxibendazole (OBZ) and ivermectin (IVM) were prepared for in vitro drug exposure, and worm viability was monitored over time. In a second drug trial, worms were used for transcriptomic analysis. The final drug concentrations employed were OBZ at 40.1 μm (10 μg mL-1) and IVM at 1.1 μm (1 μg mL-1) for 24 and 3 h, respectively. The RNA-seq analysis revealed numerous differentially expressed genes, with some being potentially related to drug detoxification and regulatory mechanisms. This report provides a method for in vitro drug exposure and the phenotypic responses for Parascaris spp., which could be extrapolated to other ascarid parasites. Finally, it also provides preliminary transcriptomic data following drug exposure as a reference point for future studies of Parascaris spp.

Dealing with double trouble: Combination deworming against double-drug resistant cyathostomins

An alternative control regimen for drug-resistant parasites is combination deworming, where two drugs with different modes of action are administered simultaneously to target the same parasite. Few studies have investigated this in equine cyathostomins. We previously reported that an oxibendazole (OBZ) and pyrantel pamoate (PYR) combination was not sustainable against a cyathostomin population with high levels of OBZ and PYR resistance. This study consisted of a field study and two computer simulations to evaluate the efficacy of a moxidectin-oxibendazole (MOX-OBZ) combination against the same cyathostomin population. In the field study, anthelmintic treatments occurred when ten horses exceeded 100 eggs per gram. Fecal egg counts and efficacy evaluations were performed every two weeks. The two simulations utilized weather data as well as equine and parasite population parameters from the field study. The first simulation repeated the treatment schedule used in the field study over a 40 year period. The second evaluated efficacies of combination treatments using selective therapy over 40 years. In the field study, efficacies of MOX and both combination treatments were 100%. The egg reappearance period for MOX was 16 weeks, and the two combination treatments were 12 and 18 weeks. The first (46.7%) and last (40.1%) OBZ efficacies were not significantly different from each other. In the simulation study, the combination treatment delayed MOX resistance development compared to when MOX was used as a single active. This occurred despite the low efficacy of OBZ. The second set of simulations identified combination treatments used with selective therapy to be the most effective at delaying MOX resistance. Overall, this study supports the use of combination treatment against drug-resistant cyathostomins, when one of the actives exhibits high efficacy, and demonstrates benefits of this approach despite substantially lowered efficacy of the other active ingredient.

Oxibendazole inhibits prostate cancer cell growth.

Prostate cancer (PCa) is one of the most common malignancies among men and is the second leading cause of cancer-associated mortality in the developed world. Androgen deprivation therapy (ADT) is the most common treatment for PCa. However, the majority of androgen-sensitive PCa patients will eventually develop resistance to ADT and the disease will become androgen-independent. There is, therefore, an immediate requirement to develop effective therapeutic techniques towards the treatment of recurrent PCa. Oxibendazole (OBZ) is an anthelmintic drug that has also shown promise in the treatment of malignancies. In the present study, the capability of OBZ to repress the growth of PCa cells was assessed in human androgen-independent PCa 22Rv1 and PC-3 cell lines. The growth of the 22Rv1 and PC-3 cell lines, as assessed with a trypan blue exclusion assay, was markedly inhibited by OBZ treatment in vitro, with half-maximal inhibitory concentration values of 0.25 and 0.64 µM, respectively. The mean size of 22Rv1 tumors in nude mice treated with OBZ (25 mg/kg/day) was 47.96% smaller than that of the control mice. Treatment with OBZ increased the expression of microRNA-204 (miR-204), as determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and the level of p53 as determined with western blotting, two well-characterized tumor suppressor genes. When miR-204 expression was knocked down by introduction of an miR-204 inhibitor, the inhibitory effect of OBZ was markedly reduced; however, when it was overexpressed, the inhibitory efficiency of OBZ was markedly higher, indicating that upregulation of miR-204 is key for the efficacy of OBZ. Additionally, OBZ was demonstrated with RT-qPCR to repress the expression of the androgen receptor, and by western blotting to reduce prostate-specific androgen in 22Rv1 cells. The results suggest that OBZ has potential for clinical use in the treatment of recurrent PCa.

Evaluation of activity of fenbendazole, oxibendazole, piperazine , and pyrantel pamoate alone and combinations against ascarids, strongyles, and strongyloides in horse foals in field tests on two farms in Central Kentucky in 2014 and 2015

Activity of fenbendazole (FBZ), oxibendazole (OBZ), piperazine (PIP) and pyrantel pamoate (PRT) alone and combinations of OBZ and PIP and of OBZ and PRT was evaluated against parascarids, strongyles and strongyloides in horse foals (n=281).This was on two farms – Farm A – mixed light-horses (n=26) and Farm B-Thoroughbreds (n=255) – in Central Kentucky in field tests in 2014 and 2015. Foals on both farms were treated one to three times each; an exception was that seven foals on Farm A were nontreated controls. Before treatment, the foals on Farm A were all weighed on scales but weights for Farm B were estimated. Evaluation of the anthelmintics was by recording the number of foals passing specific types of nematode eggs before and after treatment using qualitative and/or quantitative (EPG) methods. Results are: 1) ascarids (parascarids) — efficacy was excellent for OBZ, PIP, OBZ-PIP, and OBZ-PRT; likewise for PRT for one group (in 2014) of Farm A foals but not another group (2015) there. FBZ was inactive against these parasites. 2) strongyles — activity was lacking or incomplete for all of the compounds alone or in combination; it was the best for OBZ-PIP, OBZ-PRT and PRT alone. 3) strongyloides — data indicated no or incomplete activity; it was highest for OBZ and OBZ-PIP. The level of drug activity against parasites in Farm B foals may in some instances have been different if exact rather than estimated body weights had been obtained before treatment like for Farm A foals.

Resistance of gastrointestinal nematodes to the most commonly used anthelmintics in sheep, cattle and horses in Spain

The objective of this study was to evaluate the status of anthelmintic resistance (AR) in ruminants and horses in Spain. The efficacy of commonly used macrocyclic lactones (MLs) – ivermectin (IVM) and moxidectin (MOX) – was measured in sheep, cattle and horses. In addition, albendazole (ABZ) and levamisole (LEV) were evaluated in sheep and oxibendazole (OXI) and pyrantel (PYR) in horses. Efficacy was evaluated based on the difference between the arithmetic mean pre- and post-treatment faecal egg count (in cattle and horses), or compared to an untreated control group (in sheep). AR was present when the percentage reduction in egg count was <95% and the lower 95% confidence interval (CI) was <90%; if only one of these two criteria was met, the finding was recorded as suspected AR (SAR). In horses, AR–PYR and OXI was considered when the percentage reduction in egg count was ≤90% and the lower 95% CI was ≤80%. For each animal species, at least 10 study sites were selected.AR to at least one of the drugs was detected in all 10 sheep flocks; the main parasite identified after treatment was Teladorsagia circumcincta. Moreover, in 5 flocks multidrug resistance was identified, on 4 farms to drugs from different families, on one farm to both MOX and IVM and on another farm to all drugs tested. In cattle, the efficacy of both MOX and IVM was 100% on 4 and 3 farms, respectively, and therefore 60% of these farms were considered to have AR or SAR to both MLs. The most frequent parasite identified after treatment was Trichostrongylus spp., although Ostertagia ostertagi was also identified after treatment on one farm. In contrast to ruminants, the 4 drugs evaluated in horses were highly efficacious against strongyles, with efficacies for the MLs and OXI between 95 and 100% and between 94 and 100% for PYR, although 3 herds were SAR against PYR.In conclusion, AR to at least one of the commonly used drugs was identified on all sheep flocks investigated in the northwest of Spain. The occurrence of AR to MLs in cattle was higher than expected but consistent with what was observed in sheep. In horses, all currently used drugs were confirmed as effective against strongyles.

A combination treatment strategy using pyrantel pamoate and oxibendazole demonstrates additive effects for controlling equine cyathostomins

SummaryAnthelmintics of the avermectin/milbemycin class continue to demonstrate the highest efficacy against cyathostomin nematodes; however, over‐reliance on these drugs is increasing the likelihood that recent emerging resistance to these drugs will worsen. Use of benzimidazole and pyrantel compounds given concurrently may provide sufficiently high efficacy to serve as a viable treatment alternative to the avermectin/milbemycin drugs, thus reducing the selective pressures for resistance development. The study was conducted to determine if oxibendazole (OBZ) and pyrantel pamoate (PYR) would have greater efficacy when used in combination vs. what either drug could achieve individually, and to determine if the combination protocol would consistently achieve faecal egg count reduction (FECR) rates &gt;90%, the general threshold for acceptable efficacy. Horses of various ages and breeds on 11 horse farms were assigned randomly to treatment with OBZ (n = 34), PYR (n = 35), or a combination of both (n = 61). A faecal egg count was performed for each horse prior to treatment and 9–14 days post treatment. Mean FECR percentages were calculated for each treatment group on individual farms, and for each treatment group across all farms. Combination treatment achieved &gt;90% mean FECR on all 11 farms, and &gt;95% on 9 of the 11 farms. Overall arithmetic mean FECR rates were 90.03%, 81.10% and 96.35% for horses treated respectively with OBZ, PYR, and the 2 drugs in combination. Combination treatment with OBZ and PYR demonstrated an additive effect whereby horses given both anthelmintics concurrently had mean FECR percentages that were significantly greater than in horses given either drug alone. Combination treatment with OBZ and PYR can be an efficacious and viable treatment choice for controlling cyathostomins, thereby reducing the over‐reliance on avermectin/milbemycin drugs.

Parasite field study in central Kentucky on thoroughbred foals (born in 2004) treated with pyrantel tartrate daily and other parasiticides periodically

Foals (79), born in 2004 on three thoroughbred horse farms (C, M, and S) in central Kentucky, were fed pyrantel tartrate daily, beginning at about 3 months of age. In addition, other parasiticides [fenbendazole (FBZ), ivermectin (IVM) alone or with praziquantel (PRAZ), oxibendazole (OBZ), pyrantel pamoate (PRT), and moxidectin (MOX)] were given periodically. All treatments were administered by farm personnel. Over a 14-month period, from May 2004 to July 2005, collections (n=989) of feces were made from the foals for determination of presence of internal parasite eggs/oocysts by qualitative and/or quantitative methods. Conclusions on drug activity are based necessarily on considering the combined effect of pyrantel tartrate and the other compounds. For small strongyles, this was related to which specific additional compound was given. Based on the percentage of foals with strongyle-egg-positive feces and/or the level of eggs per gram of feces (EPG) counts for the foals after treatment, drug activity on small strongyles was highest to lowest for MOX, IVM and IVM/PRAZ, FBZ, OBZ, PRT, and FBZ (2x for 5 days). The macrocyclic lactones (MOX and IVM) were highly superior to the other compounds. Some of the strongyle counts were high (over 2,000), especially on one farm (S), during periods when foals received only pyrantel tartrate, but a few days after administration of therapeutic dose rates of the drugs IVM or MOX, they were negative or very low. Ascarid eggs were present in feces of three foals after treatment with a combination of IVM and PRAZ. The qualitative method was more efficient than the quantitative method in detection of ascarid and strongyle eggs in the feces. Prevalence of eggs of ascarids (Parascaris equorum) was low (0, 4, and 31%), of strongyles high (80, 100, and 100%), of Strongyloides westeri very low (only one infected foal), and oocysts of Eimeria leuckarti medium to high (36, 41, and 85%) for the three farms, C, M, and S, respectively. It is uncertain whether the low ascarid prevalence was from activity of pyrantel tartrate and/or the other drugs or to a limited source of infective eggs.

Field studies on endoparasites of Thoroughbred foals on seven farms in central Kentucky in 2004

Fecal samples (n=1,584) for a parasite study were collected from Thoroughbred foals (n=349), 28 to 330 days old, on seven farms in central Kentucky during 49 monthly trips from May to October, November, or December 2004. The main purpose was to determine possible drug resistance of ascarids, also of strongyles, to ivermectin (IVM) and other commercially available compounds [fenbendazole, moxidectin (MOX), oxibendazole (OBZ), and pyrantel pamoate]. In addition, interest was on prevalence of foal parasites. Qualitative data were obtained by recording presence or absence of ascarid and strongyle eggs in feces from 7 to 25 days after treatment of some of the foals. None of the compounds completely eliminated ascarid eggs in feces of all foals posttreatment. Activity on ascarids was significantly the highest for OBZ and lowest for IVM. Activity on strongyles was significantly higher for IVM and MOX than for the other compounds. Prevalence (mean percentage of foals infected) was recorded for eggs of Parascaris equorum (39%), of strongyles (32%), and of Strongyloides westeri (2%) and oocysts of Eimeria leuckarti (28%) in feces of foals. One or more foals had infections of P. equorum on six farms (86%), strongyles on seven farms (100%), S. westeri on two farms (29%), and E. leuckarti on six farms (86%). The oldest foal infected with E. leuckarti was 301 days of age. Comparison was made on the prevalence of parasites in foals on five of the same farms in the 2004 study, which were part of a similar 14-farm project in 2003 (Lyons and Tolliver in Parasitol Res (2004) 92:400-404).

Quantitative chromatographic determination of several benzimidazole anthelmintic molecules in parasite material

A specific, precise and accurate high-performance liquid chromatographic (HPLC) analytical method has been developed for the quantitative determination of different benzimidazole (BZD) anthelmintics in parasite material ( Moniezia benedeni). Mebendazole (MBZ), oxibendazole (OBZ), flubendazole (FLBZ), albendazole (ABZ) ricobendazole (RBZ), albendazole sulphone (ABZSO 2), fenbendazole (FBZ), oxfendazole (OFZ) and fenbendazole sulphone (FBZSO 2) were measured simultaneously in M. benedeni, a sheep and cattle cestode parasite used as a model of the biological matrix. The recovery, linearity, precision, accuracy, limits of detection and quantification of the method were determined. Drug extraction from the parasite’s tissue homogenate was performed using methanol (liquid phase extraction), and after solvent evaporation, the residual material was cleaned up by solid phase extraction prior to analysis by reversed-phase HPLC. The resolution of all the BZD molecules assayed was obtained on a C 18 reversed-phase (5 μm, 250 mm×4.6 mm) column using acetonitrile and ammonium acetate as the mobile phase and ultraviolet (UV) detection at 292 nm. Regression analyses for all the BZD compounds assayed were linear at concentrations ranging from 1.61 to 64.21 nmol/100 mg protein (triplicate determinations) showing correlation coefficients greater than 0.9922. The developed method is efficient for the simultaneous determination of several benzimidazole anthelmintic molecules in parasite material and useful for the ex vivo and in vivo characterisation of the kinetics of drug uptake/diffusion in target parasites, which seems to be relevant to optimise parasite control both in human and veterinary medicine.

Population-S benzimidazole- and tetrahydropyrimidine-resistant small strongyles in a pony herd in Kentucky (1977-1999): effects of anthelmintic treatment on the parasites as determined in critical tests.

Population-S small strongyles have been studied since 1974 in central Kentucky in a closed Shetland pony breeding herd. The ponies were treated approximately every 8 weeks with cambendazole (1974-1978), oxibendazole (OBZ) (1978-1992), or pyrantel pamoate (PRT) (1992-1999). Small strongyles in the ponies have shown resistance to these compounds in field and critical tests. One purpose of this presentation was to compare different parameters for determination of effects on the small strongyle species in ponies after treatment, mainly with OBZ or PRT, from data in critical tests (n=112). Also, the objective was to report on relative changes in the composition of species of small strongyles during the period 1977 through 1999. The following entities were compared to evaluate the effect of OBZ- or PRT-treatment on the small strongyles: (1) numbers of specimens with eggs in utero--there were less gravid worms passed in the feces than recovered at necropsy for OBZ but the numbers of gravid worms were similar in both categories for PRT, (2) pre- and posttreatment counts of eggs per gram of feces (EPGs) and larvae per gram of feces (LPGs)--the reductions were greater for the counts of EPGs than LPGs for OBZ but not for PRT, and (3) pre- and posttreatment counts of EPGs versus % of worms removed--reductions of the former were greater than the latter for both compounds. As shown from data in this study, reduction of EPG counts post treatment indicated much greater drug activity than was actually demonstrated by removal of worms. One evident factor was the value of doing critical tests to verify posttreatment counts of EPGs as indicators of anthelmintic activity. Twenty-eight species of small strongyles were found. For the 20 most prevalent species in the study, two decreased, five remained unchanged, three increased and then became stationary, five increased but then decreased, and five increased progressively. Numbers of small strongyles were highest in 1987 and 1999.

Transtegumental diffusion of benzimidazole anthelmintics into Moniezia benedeni: correlation with their octanol–water partition coefficients

The experiments described here report on the correlation between the ex vivo diffusion of different benzimidazole (BZD) anthelmintics into the cestode parasite Moniezia benedeni, and their octanol–water partition coefficients (P.C.). The characterisation of the drug diffusion process into target parasites is relevant to understand the mechanism of drug penetration and the pharmacological activity of anthelmintic drugs. Specimens of the tapeworm M. benedeni, used as a helminth parasite model, were obtained from untreated cattle killed at the local abattoir. The collected parasites were incubated (5–210 min) with either fenbendazole (FBZ), albendazole (ABZ), ricobendazole (RBZ), oxfendazole (OFZ), mebendazole (MBZ), oxibendazole (OBZ), or thiabendazole (TBZ), in a Kreb’s Ringer Tris buffer medium at a final concentration of 5 nmol/ml. After the incubation time elapsed, samples of parasite material were chemically extracted and prepared for high performance liquid chromatography (HPLC) analysis to measure drug/metabolite concentrations. Additionally, the octanol–water P.C. for each molecule was estimated as an indicator of drug lipophilicity, using reversed phase HPLC analysis. All the incubated drugs were recovered from the tapeworms as early as 5 min post incubation. There was a high correlation ( r=0.87) between drug lipophilicity, expressed as octanol–water P.C. (Log P), and drug availability within the parasite. The most lipophilic BZD compounds (FBZ, ABZ, and MBZ), with P.C. values higher than 3.7, were measured at significative higher concentrations within the tapeworm compared to those drugs with the lowest P.C. values. Considering the results from the current and previous studies, it is clear that passive diffusion is a major mechanism of BZD penetration into cestode parasites, where lipid solubility is a determinant factor influencing the diffusion of these anthelmintic molecules through the parasite tegument. Index Descriptors and Abbreviations: Transtegumental diffusion; Benzimidazole anthelmintics; Cestodes; Moniezia benedeni; Lipophilicity; Octanol–water partition coefficient

Evaluation of tests for anthelmintic resistance in cyathostomes

Resistance, especially to the anthelmintic benzimidazoles (BZ), has been reported in horse cyathostomes world-wide. Diagnosis of resistance has traditionally been made by faecal egg count reduction (FECR) trials, however, this technique has limitations. Some of the shortcomings may be resolved by refining the test or by using an in vitro test. FECR tests and the larval development assay (LDA) were performed on adult horses held on 15 different horse properties across a wide geographical area of NSW, Australia. FECR were measured before and 10–14 after days treatment with oxibendazole (OBZ), morantel (MOR) or ivermectin (IVM) at recommended dose rates. Eight properties were rejected following low pre-treatment egg counts, leaving seven in the study. On these, the majority of larvae recovered from faecal cultures were cyathostomes. Using a definition of resistance as a FECR of <90%, resistance to OBZ was present on six properties and to MOR on two properties. Resistance to IVM was not detected. An alternative method of calculating FECR based on individual horse egg counts pre- and post-treatment was developed and results from the same properties compared with the results of the LDA. For example, for the BZ, correlation coefficients of values of lethal concentration to kill 50% of population (LC 50) on LDA and FECR percentages were −0.536 before and −0.704 after OBZ treatment. We conclude that the LDA has the potential to be a single visit test for detection of anthelmintic resistance in horse cyathostomes but requires further investigation and standardisation.

Continuance of studies on Population S benzimidazole-resistant small strongyles in a Shetland pony herd in Kentucky: effect of pyrantel pamoate (1992–1999)

Research on benzimidazole-resistant Population S small strongyles began in a Shetland pony herd in 1974 at the University of Kentucky and has continued for over 25 years. The present update, for the period 1992–1999, evaluated activity of pyrantel pamoate (PRT) in field tests in the pony herd. Additional critical tests with PRT and oxibendazole (OBZ) were done in foals born in the herd. Activity of PRT was initially excellent in field tests, based on epg/lpg count data, but declined rapidly during the second full year of pyrantel treatments. Critical test data for small strongyles indicated efficacies of PRT were about 60% at the beginning of the present observations and this intermediate level of removal continued throughout the seven-year period except for 1994 (75%). Unusual was the finding that field test epg/lpg data on small strongyles indicated much lower activity of PRT than found in worm count data in critical tests. The previously reported ineffective activity of OBZ on this population of small strongyles continued. Data are presented on prevalence and drug activity on several species of internal parasites besides small strongyles.

Toxocara canis infection in the paratenic host: a study on the chemosusceptibility of the somatic larvae in mice

A research on formulation and dosage strategies of anthelmintics have been conducted in mice experimentally infected with eggs of Toxocara canis. Multidose treatments commenced at days 2, 14, 81, 87 and 123 postinfection. Results indicated no detectable difference in the chemosusceptibility of the migrating early infection larvae and the resting hypobiotic chronic infection larvae. Application of medicated dry food (pellets) may be a simple way of improving efficacy of treatment compared to oral drenching. The larvicidal potential of fenbendazole (FBZ), albendazole (ABZ), flubendazole (FUBZ), oxibendazole (OBZ) and ivermectin was assessed. Reductions of 84.2 to 99.7 or 88.8 to 100% of group mean larval counts were recorded after 20–30-day courses of feeding pellets containing FBZ at 6 g kg −1 or ABZ at 1.6 g kg −1 food, respectively. Efficacies of 57.8 to 88.2 or 81.1 to 32.0% were achieved by 20-day courses of feeding pellets medicated with FUBZ and OBZ at 1.6 g kg −1 or 6.0 g kg −1 food, respectively. Ivermectin at various dosing regimens showed only moderate larvicidal potential. Efficacy rates were not closely correlated with the amount of drug taken by the animals. The blood–brain barrier is permeable for the anthelmintics tested, and the brain of mice does not provide a site promoting survival of larvae.

Experimental cyathostome challenge of ponies maintained with or without benefit of daily pyrantel tartrate feed additive: comparison of parasite burdens, immunity and colonic pathology

Eighteen mixed-breed, naturally infected ponies ranging in age from 1 to 16 yr and four cyathostome-naı̈ve ponies reared and maintained under parasite-free conditions ranging in age from 1 to 4 yr were used in this study. Naturally-infected ponies were treated with 1 dose of ivermectin (IVM) at 200 μg kg −1, followed by a 5-day regimen of oxibendazole (OBZ) at 20 mg kg −1 to remove existing cyathostome burdens; cyathostome-naı̈ve control ponies were treated with IVM alone. The naturally infected ponies were matched on age and gender, then randomly assigned to one of three treatment groups of six animals per group; the four cyathostome-naı̈ve ponies constituted a fourth group. Following OBZ treatment, Group 1 ponies were treated with pyrantel tartrate (PT) in their pelleted ration; the remaining ponies received only the pelleted ration. Beginning on experiment Day 3, a daily challenge infection of 10 4 mixed cyathostome larvae was administered orally to ponies of Group 1, Group 2 and the cyathostome-naı̈ve controls. Group 3 ponies served as unchallenged controls to determine residual parasite burdens following IVM/OBZ treatment. Necropsy examinations were performed on three Group 3 ponies on Day 1; the remainder of the necropsy examinations began on Day 41. Cyathostome burdens were evaluated by recovery of larvae and adults from the luminal contents, by digestions of the intestinal mucosa, and by mural transillumination of full-thickness intestinal sections. Differences in postchallenge clinical responses were also compared. Necropsy examinations included comparisons of grossly visible inflammation of the large bowel, weights of biopsy specimens from each region, and histologic evaluations of these biopsies. Parasite recoveries at necropsy indicated a strong protective effect derived from daily PT treatment. Mean weights of intestinal biopsies corresponded with worm burdens, but histological evaluation did not reveal architectural or cellular changes to account for the increase in weight; therefore, edema was suspected. A strong age-related resistance to challenge infection was apparent in both the PT-treated and control groups by virtue of the lower mean worm burdens found in older ponies compared to younger ponies of the same treatment group; however, daily PT treatment of older ponies reduced the variability of their worm burdens to a uniformly low level. Comparisons of luminal and mucosal parasite burdens of age stratified nontreated controls further suggest that the age related resistance, which is acquired, targets increasing numbers of parasite stages as this resistance matures. Further, there is no evidence for an immune mediated acquisition of hypobiotic L 3.

Identification and characterization of a pyrantel pamoate resistant cyathostome population

Three fecal egg count reduction assays (FECR) and one critical trial were performed to determine the efficacy of pyrantel pamoate (PP) at 6.6 mg base kg −1 on a well managed stud farm in Louisiana where a loss of efficacy was suspected. Efficacy of PP based on FECR varied from 25% in mares to 83% in yearlings. Second treatments with PP 2 weeks following an initial treatment failed to reduce eggs per gram (EPG). A critical trial was performed to determine the cyathostome species resistant to PP. Three strongyle-naive ponies which acquired infections on the farm were used for this purpose. Following treatment with PP at the recommended dose, 11 species of cyathostomes remained in the intestine of the tracer ponies. Reduced efficacies (62%–88%) were noted for seven species. Resistance to oxibendazole (OBZ), which was > 90% effective on this farm in 1982, was also evaluated by FECR and found to exist. The results of one experiment indicate that dual resistance of parasites to PP and OBZ also exists.

A study (1977–1992) of population dynamics of endoparasites featuring benzimidazole-resistant small strongyles (Population S) in Shetland ponies

Critical tests (91) were done between 1977 and 1992 in Shetland ponies to evaluate drug susceptibility and population dynamics (present paper) of endoparasites. The test ponies, most less than 1 year old, were from a herd where older animals were treated every 8 weeks initially with cambendazole (CBZ) (1974–1978) and then with oxibendazole (OBZ) (1978–1992). Previous field test data (1974–1992) on older ponies in the breeding herd indicated the presence of benzimidazole (BZ) resistant small strongyles. Data on population dynamics from the present critical tests indicated that 28 species of small strongyles persisted over the study period in spite of initial susceptibility and later refractiveness of six species to both CBZ and OBZ. Changes in intensities and other aspects were observed for the six BZ-resistant species ( Cyathostomum catinatum, Cyathostomum coronatum, Cylicocyclus nassatus, Cylicostephanus calicatus, Cylicostephanus goldi, and Cylicostephanus longibursatus). Variabilities, some striking, were found in prevalence and intensity in bots, stomach worms, ascarids, eyeworms, large strongyles, pinworms and tapeworms.

Critical test evaluation (1977–1992) of drug efficacy against endoparasites featuring benzimidazole-resistant small strongyles (Population S) in Shetland ponies

Several compounds ( n = 13 single or combinations; most at therapeutic dosages) were evaluated between 1977 and 1992 in critical tests ( n = 91) against benzimidazole (BZ) resistant small strongyles (Population S) and several other species of internal parasites in Shetland ponies, mostly under 1 year old. The closed breeding herd, from which the test ponies were selected, had been treated every 8 weeks with cambendazole (CBZ) for 4 years (1974–1978) and oxibendazole (OBZ) for 14 years (1978–1992). Published field test data (1974–1992) on older ponies in the herd showed BZ resistance of small strongyles. Average efficacies in the present critical tests against small strongyles for OBZ ( n = 59 animals) were high in early years (95% or higher), but gradually declined to a low of 1% in 1991. Side-resistance of small strongyles was evident in critical tests ( n = 1–6/single drug or combination) for several other BZs and a pro-BZ; ivermectin and piperazine were highly active, but pyrantel pamoate exhibited weak activity. BZ resistance was evident for six small strongyle species ( Cyathostomum catinatum, Cyathostomum coronatum, Cylicocyclus nassatus, Cylicostephanus calicatus, Cylicostephanus goldi, and Cylicostephanus longibursatus). Activity on bots, ascarids, large strongyles, and pinworms was essentially as expected, indicating no drug resistance.

Ion-Pair Isolation and Liquid Chromatographic Determination of Albendazole, Oxfendazole, Oxibendazole, and Thiabendazole Residues in Milk

Abstract A liquid chromatographic [HPLC] method for the determination of Albendazole (ABZ), Oxfendazole (OFZ), Oxibendazole (OXBZ), and Thiabendazole (TBZ) residues in milk at levels below 3 ppb has been developed. Samples were deproteinized with acetonitrile, defatted with hexane partition, and evaporated to constant volume. Following addition of acidified aqueous octane-1-sulfonate solution, the target benzimidazoles were selectively extracted as ion pairs into chloroform and analyzed on a reversed-phase C18, 5 μm, column. Overall recoveries were found to be 93.5 ± 2.6% for OFZ, 90.0 ± 1.3% for OXBZ, 85.7 ± 3% for ABZ, and 74.0 ± 2.6% for TBZ. Limited data on the depletion of OXBZ and ABZ in sheep milk were also generated.